Project Summary and Abstract Asthma is a chronic inflammatory disease characterized by inflammation, mucus production, airway remodeling, and hyper-responsiveness resulting in severe bronchoconstriction. These processes involve interplay among resident airway cells and infiltrated inflammatory cells. Allergic inflammatory mediators such as cytokines, chemokines, and growth factors act on resident airway cells including airway smooth muscle (ASM) resulting in structural and functional changes. However, knowledge gaps remain in our understanding of the molecular factors and mechanisms by which inflammatory mediators modulate ASM phenotype. Inflammatory mediators change gene expression and contribute to the pathogenesis of asthma. Transcriptional regulation of gene expression in resident airway cells has been studied extensively. However, protein function in a target cell can be regulated at multiple levels starting from transcription followed by post-transcription, translation and post-translation steps. In this context, small non-coding RNAs (ncRNAs) including the ones derived from transfer RNA (tRNA) have evolved as one of the key regulators of gene expression post-transcriptionally. We propose that tRNA halves, a major subgroup of tRNA-derived ncRNAs, play important roles in asthma pathobiology and can be targeted as asthma therapy. In preliminary studies, we found that mouse lung expresses specific tRNA half species whose levels are significantly upregulated during allergic inflammation caused by inhaled challenge of house dust mite (HDM). Furthermore, human ASM cells express tRNA halves and their expressions are promoted by treatments with pro-inflammatory and Th2 cytokines, tumor necrosis factor alpha (TNF?) and interleukin 13 (IL-13). These results allow us to define the tRNA halves as a novel class of cytokine-dependent tRNA halves, termed proinflammatory tRNA-derived RNAs (pit-RNAs). Importantly, siRNA-directed depletion of pit-RNAs significantly impaired the proliferation of ASM cells, suggesting that pit-RNAs are not just accumulated as degradation by-products of tRNAs but are expressed as functional RNAs promoting ASM cell growth. These results have led us to hypothesize that inflammatory mediators accumulate pit-RNAs in ASM cells, which enhance ASM growth in the molecular pathogenesis of asthma. We propose to comprehensively identify pit-RNA expression profiles (Aim 1) and investigate molecular mechanisms of the pit-RNA-mediated modulation of ASM cell function (Aim 2), which will reveal a novel tRNA-engaged small RNA pathway in the pathogenesis of asthma and support the exploration of biomarkers and efficacious therapeutic applications targeting tRNA halves. .